The eukaryotic plasma membrane forms a crucial interface between the cell and the external milieu. Rather than a simple homogeneous mixture of components, the plasma membrane is a complex dynamic heterogeneous structure. The origins of this heterogeneity are diverse and include several thousand species of lipid, compartmentalization by a sub-membrane actin cytoskeleton and the lateral assembly of sphingolipids and cholesterol. The overall aim of this research program is to dissect the function and composition of specific plasma membrane assemblies, including Ras signaling domains and caveolae, herein referred to as nanodomains. Caveolae are cell surface pits which are an abundant feature of mammalian cells. Caveolae have been implicated in signal transduction, lipid regulation, endocytosis, and mechanosensation. Caveolae dysfunction has been linked to cell transformation and to muscle disease. Ras proteins operate as molecular switches in many signal transduction pathways and are frequently mutated in human tumors. Different Ras isoforms occupy, distinct, non-overlapping nanodomains of the cell surface. The aims of this project are to map structural components of Ras nanodomains and caveolae that are involved in the formation and function of these domains. The dynamic association of Ras isoforms with specific plasma membrane nanodomains will be dissected using a combination of novel electron and light microscopic methods, biochemical techniques, functional assays and by modeling in silico. The molecular mechanisms involved in caveolae formation will also be investigated. Newly identified components implicated in caveolae formation will be characterized in detail in order to understand how these components contribute to caveolae formation and caveolae function. These studies will provide new insights into fundamental aspects of membrane organization in eukaryotic cells and will lead to a new molecular understanding of the plasma membrane, relevant to numerous human diseases. PUBLIC HEALTH RELEVANCE This project seeks to understand how the surface of cells is organized at the molecular level. The proposed research will provide new insights into the way in which proteins and lipids come together to generate assemblies that have a crucial role in vital cellular processes such as the regulation of cell growth, a process perturbed in cancer.